Prostaglandins are biologically important metabolites derived from arachidonic acid. FIG. 1 shows schematically the biochemical pathways of arachidonic acid metabolism, indicating the position of the various prostaglandins (PG). As exemplified by the two structures in FIGS. 2A and 2B, prostaglandins are 20-carbon fatty acids that contain a 5-carbon ring. While structurally similar, the molecules are functionally quite diverse. Prostaglandins act as mediators in a large number of physiological processes, including hemostasis and thrombosis, and contribute to pathologic processes associated with inflammation, atherosclerosis, and bronchoconstriction. There is therefore a great deal of interest in elucidating their roles, a process that requires sensitive and specific detection at nanomolar levels in complex biological matrices.
Gas chromatography-mass spectrometry (GC-MS) has been the traditional tool for detecting metabolites in the arachidonic acid pathway. However, these methods require extensive sample preparation and cumbersome derivatization procedures. Several analytical steps are required for extraction, separation, and purification before derivatization and separation by GC-MS. While these techniques have been improved in recent years, they remain costly and laborious and yield variable results. In addition, arachidonyl-derived lipids in biological fluids, particularly plasma, are known to be relatively unstable and undergo a variety of transformations when subjected to harsh derivatization conditions. The samples therefore need to be treated carefully, and antioxidants are commonly used to prevent further oxidation.
Recently, liquid chromatographic techniques have been developed to separate prostaglandin-containing mixtures with minimal sample preparation prior to analysis. When combined with electrospray ionization (ESI) mass spectrometry, LC has picogram detection limits, which is sufficient bioanalytical sensitivity for many applications. Furthermore, MS and tandem MS can often provide necessary structure elucidation to resolve co-eluting species without tedious derivatization and sample manipulation. For example, a method for high performance liquid chromatography/tandem mass spectrometry of F2-isoprostanes is disclosed in H. Li et al., “Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine,” Proc. Natl. Acad. Sci. 96, 1999: 13381-13386. While this method is useful for the particular species studied, it cannot be generalized to all prostaglandins. One of the challenges in combining LC and ESI-MS for analyzing prostaglandins is that optimal conditions for one technique are often not ideal for the other. That is, conditions that maximize ionization efficiencies reduce chromatographic separation resolution, while ideal chromatographic conditions lead to poor electrospray ionization efficiencies.
This problem is particularly pronounced for the two prostaglandin isomers illustrated in FIGS. 2A and 2B. Prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) are isomers having different roles in inflammatory processes. PGD2 is the major eicosanoid product of mast cells and is released during allergic or asthmatic anaphylaxis, while PGE2 activates inflammatory processes and is important in fertility and gastric mucosal integrity. Because of these different functions, it is desirable for researchers to be able to distinguish and quantify the two isomers by LC-MS. For sufficient ionization of the two species, particularly at low concentrations or small sample size, negative ion mode is required, which entails basic solution conditions. Under these conditions, however, the species tend to co-elute from the chromatographic column. Because the two prostaglandin structures are so similar, differing only in the reversed positions of a hydroxyl and carbonyl group, their mass spectra cannot distinguish the co-eluted species. Furthermore, while it is often common to distinguish isomers by their tandem mass spectra (further fragmentation of the parent and subsequent ions), MS2 and MS3 tandem mass spectra of the two species are also virtually identical.
There is a need, therefore, for a LC-MS method for detecting and distinguishing between prostaglandin isomers at low concentrations. It is desirable that the method require little sample preparation and no sample derivatization and be able to detect and distinguish between picogram quantities of different prostaglandins.